A tight balance between synaptic vesicle exocytosis and endocytosis is fundamental to maintaining synaptic structure and function. As we reported recently, voltage-gated Ca2+ channels (VGCCs) are not only an integral part of the synaptic vesicle (SV) release machinery but also an essential element of SV endocytosis machinery. VGCCs and endophilin (endo), a key regulator of clathrin-mediated vesicle endocytosis, form a macromolecular complex that serves to recruit the endocytic machinery into the nerve terminal. Of particular interest is the finding that formation of the endo-channel complex is Ca2+-dependent. The effects of Ca2+ are mediated by a novel Ca2+ sensor that resides within endo. Binding of Ca2+ to endo changes its conformation from the open mode to the closed mode, thus providing a mechanism for Ca2+ to regulate SV endocytosis. The long term goal is to understand the unique role of VGCCs and their novel partner proteins, which we identified in yeast two-hybrid screening, in regulating SV release and recycling. In this application, we will focus on the interaction between VGCCs and endophilin in SV endocytosis. We will test several hypotheses predicted from our model using the combined approaches of biochemistry, molecular biology, fluorescence imaging, electrophysiology and X-ray crystallography. We will address: (1) the molecular composition of the novel Ca2+ sensor in endophilin; (2) biochemical characterization of the Ca2+ channel-endophilin-dynamin complex; (3) the effects of Ca2+ on the endophilin-channel interaction in vivo; and (4) the effects of endophilin binding on the Ca2+ channel functions and the effects of modus switching of endophilin on clathrin-mediated endocytosis. The results will advance our knowledge of the unique role of VGCCs and Ca2+ influx through VGCCs in coupling and coordinating SV exocytosis and endocytosis. On a broader scale, the results will shed light on how the cellular signaling network, established via protein-protein interactions, achieves its specificity and how protein-protein interactions can be regulated by physiological factors such as Ca2+.